CN110804609A - Whole blood RNA rapid lysis solution and application - Google Patents
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Abstract
The invention provides a whole blood RNA rapid lysate and RNA extraction method without need of first cracking erythrocytes, the method greatly reduces the use of toxic reagents, simplifies the RNA extraction step, and the RNA obtained by purification has good integrity and high purity, and can be directly used for various molecular biology experiments such as RT-PCR, Northern blot, Dot blot and the like; the invention discloses a whole blood RNA rapid lysis solution, wherein each 1000ml of the lysis solution comprises the following components in parts by weight: guanidine thiocyanate, 0.2M-4M; ammonium thiocyanate, 0.2M-3M; anhydrous sodium acetate, 0.01M-0.2M; glacial acetic acid, 0.05-0.3M; 0.1-2% (W/V) of sodium lauroyl sarcosinate; sodium chloride, 0.02M-0.3M; 1% -15% (V/V) of glycerol; water saturated phenol solution, 10% -75% (V/V); the rest is DEPC treated water.
Description
Technical Field
The invention relates to the technical field of blood RNA extraction, in particular to a whole blood RNA rapid lysate and application thereof.
Background
Blood is the most used in clinical aids because of its readily available nature. Blood tests are not only the primary basis for diagnosing various hematological disorders, but also provide a lot of important information for the diagnosis and identification of other systemic disorders. RNA extraction is a fundamental content in molecular biology research and is the basis for gene expression analysis. The rapid extraction of whole blood RNA saves researchers engaged in clinical research time, reduces the probability of RNA degradation, and can accelerate rapid diagnosis of certain diseases.
The main methods for extracting RNA from blood include precipitation, magnetic bead method and silica gel column method, depending on the type of RNA adsorption medium. The precipitation method is a method of subjecting RNA to centrifugal precipitation by utilizing the property that RNA is insoluble in ethanol and isopropanol. The method has the advantages of complicated operation steps, long time consumption, low flux and unstable quality of the obtained RNA. The basic principle of the magnetic bead method is the same as that of the silica gel column method, but the magnetic bead method enriches RNA on magnetic beads under the action of separating liquid salt and an external magnetic field, so that separation is realized. The silica gel column method is to enrich RNA on a solid phase silica gel column under the action of a separation liquid salt and realize separation through centrifugation.
The extraction method is classified into various methods such as Trizol method (phenol method), SDS method, CTAB method, guanidinium thiocyanate method, and the like, and a method using protease K in combination.
The Trizol method is good in universality and high in RNA extraction quality, and is commonly used for blood RNA extraction; however, chloroform used in the procedure is a toxic reagent, which is not good for the health of the laboratory operator. The method effectively avoids the toxic reagent, and the extraction effect is not inferior to that of the Trizol method. In other various cell lysis methods, red blood cells need to be lysed first, and then the white blood cells are obtained and then lysed. The lysis solution does not need to firstly lyse red blood cells, and can effectively shorten the RNA extraction time.
The basic principle of the current column method for extracting RNA is as follows: RNA is released from cells under the actions of sample pretreatment and lysate lysis; transferring the supernatant containing RNA to a nucleic acid purification column in a solution with high salt and low pH, and binding the RNA to the nucleic acid purification column after centrifugation; further removing impurities such as protein in the nucleic acid purification column membrane under the action of the washing solution 1; under the condition of washing liquid 2, further removing salt ions in the nucleic acid purification column membrane; finally, the RNA is eluted from the purification column under low salt conditions.
The invention provides the whole blood RNA rapid lysate and the RNA extraction method without cracking erythrocytes first, which simplify the experimental steps, shorten the whole RNA extraction time and greatly reduce the use of toxic reagents.
Disclosure of Invention
In order to achieve the purpose, the invention provides the following technical scheme: a whole blood RNA rapid lysis solution, each 1000ml volume of which is composed of the following components by mass/volume:
guanidine thiocyanate, 0.2M-4M;
ammonium thiocyanate, 0.2M-3M;
anhydrous sodium acetate, 0.01M-0.2M;
glacial acetic acid, 0.05-0.3M;
0.1-2% (W/V) of sodium lauroyl sarcosinate;
sodium chloride, 0.02M-0.3M;
1% -15% (V/V) of glycerol;
water saturated phenol solution, 10% -75% (V/V);
the rest is DEPC treated water.
The application of the whole blood RNA rapid lysate in the whole blood column method for extracting RNA;
the method comprises the following steps:
step one, whole blood lysis: adding 1ml of the rapid lysis solution into a 2ml centrifuge tube, adding 500 mu l of fresh whole blood or bone marrow, covering a tube cap, carrying out vortex oscillation for 30 seconds, and centrifuging at 13,000rpm for 10 minutes;
step two, passing the RNA-containing supernatant through a purification column: adding 400 mu l of absolute ethyl alcohol into a new 1.5ml centrifuge tube, sucking 700 mu l of the centrifugal supernatant obtained in the first step, transferring the centrifugal supernatant into the tube, and uniformly blowing and sucking the centrifugal supernatant by using a suction head; pipetting 600. mu.l of the mixture, transferring the mixture to a nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute; discarding the filtrate, placing the nucleic acid purification column back into a 2ml centrifuge tube, transferring the remaining mixture to the nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute;
step three, washing and deproteinizing: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 500. mu.l of washing solution 1, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step four, washing and desalting: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 700. mu.l of washing solution 2, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step five, eluting RNA: discarding the filtrate, putting the nucleic acid purification column back into a 2ml centrifuge tube, and centrifuging at 14,000rpm for 1 minute; discarding the filtrate and centrifuge tube, placing the nucleic acid purification column into a 1.5ml of centrifuge tube without RNase, adding 50 μ l of RNase-Free Water into the center of the membrane of the purification column, tightly covering the tube cover, standing at room temperature for 1 minute, and centrifuging at 12,000rpm for 30 seconds; the column was discarded and the eluted RNA was used immediately for molecular biology experiments or stored at-80 ℃ until use.
Preferably, the nucleic acid purification column is coated with SiO2Silica gel adsorption column of the material.
Preferably, the washing solution 1 is prepared from guanidine thiocyanate 0.5M-4M, Tris-HCl (pH6.0-pH8.0)10mM-100mM, EDTA-2Na (pH8.0)10mM-50mM and absolute ethyl alcohol 40% -60%.
Preferably, the washing solution 2 is 10mM-100mM of Tris-HCl (pH6.0-pH8.0), 20mM-300mM of sodium chloride, and 60% -80% of absolute ethyl alcohol.
Preferably, the RNase-Free Water is 0.1% DEPC treated Water.
The invention has the beneficial effects that: the rapid lysate of the invention does not need to separate the white blood cells in advance, greatly shortens the extraction time of the whole blood RNA, and has the characteristics of rapidness, conciseness and high efficiency; the RNA obtained by purification has good integrity and high purity, and can be directly used for various molecular biological experiments such as RT-PCR, Northern blot, Dot blot and the like.
The whole blood sample can be fresh whole blood or frozen whole blood at-80 ℃, the subsequent operation can be suspended after the whole blood is cracked, the cracked whole blood is directly frozen at-20 ℃ or-80 ℃, and the integrity and yield of RNA are not influenced when the experiment operation is subsequently carried out.
Drawings
In FIG. 1, left 1 and right 1 are graphs showing the delamination of human anticoagulated whole blood after the human anticoagulated whole blood is lysed with the lysis solution of example 1 and example 2, respectively, and centrifuged at 13,000rpm for 10 minutes.
FIG. 2 is an electrophoretic test image of RNA extraction from 4 portions of fresh human anticoagulated whole blood using the formulation of example 1 of the present invention; wherein lane M is DL 2,000 DNAmarker.
FIG. 3 is an electrophoretic image of RNA extraction from anticoagulated whole blood stored at-80 ℃ for one month using the method of example 3 of the present invention; wherein lane M is DL 2,000 DNAmarker.
FIG. 4 is an electrophoretic image of RNA obtained by performing rapid lysis of 3 portions of fresh anticoagulated whole blood, storing the supernatant at-20 ℃ for one week, and performing a subsequent extraction step, according to example 4 of the present invention.
Detailed Description
Example 1
A preparation method of a rapid whole blood RNA lysate, wherein each 1000ml of the lysate consists of the following components in mass/volume:
guanidine thiocyanate, 236.32g
Ammonium thiocyanate, 60.89g
Anhydrous sodium acetate, 4.102g
Glacial acetic acid, 14.25ml
Sodium lauroyl sarcosinate, 2.5g
Sodium chloride, 11.68g
Glycerol, 75ml
Aqueous saturated phenol solution, 500ml
The rest is DEPC treated water.
4 parts of 500. mu.l human anticoagulated whole blood are subjected to rapid lysis and RNA extraction, comprising the following steps:
step one, whole blood lysis: adding 1ml of the rapid lysis solution into a 2ml centrifuge tube, adding 500 mu l of fresh human anticoagulated whole blood, covering a tube cover, carrying out vortex oscillation for 30 seconds, and centrifuging at 13,000rpm for 10 minutes; taking a photograph of 4 parts with consistent layering conditions, as shown in the left 1 of the figure 1;
step two, passing the RNA-containing supernatant through a purification column: adding 400 mu l of absolute ethyl alcohol into a new 1.5ml centrifuge tube, sucking 700 mu l of the centrifugal supernatant obtained in the first step, transferring the centrifugal supernatant into the tube, and uniformly blowing and sucking the centrifugal supernatant by using a suction head; pipetting 600. mu.l of the mixture, transferring the mixture to a nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute; discarding the filtrate, placing the nucleic acid purification column back into a 2ml centrifuge tube, transferring the remaining mixture to the nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute;
step three, washing and deproteinizing: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 500. mu.l of washing solution 1, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step four, washing and desalting: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 700. mu.l of washing solution 2, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step five, eluting RNA: discarding the filtrate, putting the nucleic acid purification column back into a 2ml centrifuge tube, and centrifuging at 14,000rpm for 1 minute; discarding the filtrate and centrifuge tube, placing the nucleic acid purification column into a 1.5ml of centrifuge tube without RNase, adding 50 μ l of RNase-Free Water into the center of the membrane of the purification column, tightly covering the tube cover, standing at room temperature for 1 minute, and centrifuging at 12,000rpm for 30 seconds; the column was discarded and the eluted RNA was used immediately for molecular biology experiments or stored at-80 ℃ until use.
Table 1 shows OD values of RNA extracted from four whole blood samples according to the protocol of example 1, measured on a multifunctional microplate reader.
TABLE 1
Example 2
A preparation method of a whole blood RNA rapid lysate, wherein each 1000ml volume of the whole blood RNA rapid lysate comprises the following components by mass/volume:
guanidine thiocyanate, 118.16g
Ammonium thiocyanate, 30.45g
Anhydrous sodium acetate, 8.20g
Glacial acetic acid, 21.5ml
Sodium lauroyl sarcosinate, 5g
Sodium chloride, 5.84g
Glycerol, 50ml
Aqueous saturated phenol solution, 300ml
The rest is DEPC treated water.
1 part of 500. mu.l of human anticoagulated whole blood is subjected to rapid lysis and RNA extraction, comprising the following steps:
step one, whole blood lysis: adding 1ml of the rapid lysis solution into a 2ml centrifuge tube, adding 500 mu l of fresh human anticoagulated whole blood, covering a tube cover, carrying out vortex oscillation for 30 seconds, and centrifuging at 13,000rpm for 10 minutes; the layering situation is shown in fig. 1, right 1;
step two, passing the RNA-containing supernatant through a purification column: adding 400 mu l of absolute ethyl alcohol into a new 1.5ml centrifuge tube, sucking 700 mu l of the centrifugal supernatant obtained in the first step, transferring the centrifugal supernatant into the tube, and uniformly blowing and sucking the centrifugal supernatant by using a suction head; pipetting 600. mu.l of the mixture, transferring the mixture to a nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute; discarding the filtrate, placing the nucleic acid purification column back into a 2ml centrifuge tube, transferring the remaining mixture to the nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute;
step three, washing and deproteinizing: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 500. mu.l of washing solution 1, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step four, washing and desalting: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 700. mu.l of washing solution 2, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step five, eluting RNA: discarding the filtrate, putting the nucleic acid purification column back into a 2ml centrifuge tube, and centrifuging at 14,000rpm for 1 minute; discarding the filtrate and centrifuge tube, placing the nucleic acid purification column into a 1.5ml of centrifuge tube without RNase, adding 50 μ l of RNase-Free Water into the center of the membrane of the purification column, tightly covering the tube cover, standing at room temperature for 1 minute, and centrifuging at 12,000rpm for 30 seconds; the column was discarded and the eluted RNA was used immediately for molecular biology experiments or stored at-80 ℃ until use.
Example 3
A preparation method of a whole blood RNA rapid lysate, wherein each 1000ml volume of the whole blood RNA rapid lysate comprises the following components by mass/volume:
guanidine thiocyanate, 236.32g
Ammonium thiocyanate, 152.24g
Anhydrous sodium acetate, 2.75g
Glacial acetic acid, 9.5ml
Sodium lauroyl sarcosinate, 2.5g
Sodium chloride, 5.84g
Glycerol, 75ml
Aqueous saturated phenol solution, 500ml
The rest is DEPC treated water.
RNA extraction was performed on 3 parts of human anticoagulated whole blood stored at-80 ℃ for one month, comprising the following steps:
step one, whole blood lysis: adding 1ml of the rapid lysis solution into a 2ml centrifuge tube, adding 500 mu l of unfrozen human anticoagulated whole blood, covering a tube cover, carrying out vortex oscillation for 30 seconds, and centrifuging at 13,000rpm for 10 minutes;
step two, passing the RNA-containing supernatant through a purification column: adding 400 mu l of absolute ethyl alcohol into a new 1.5ml centrifuge tube, sucking 700 mu l of the centrifugal supernatant obtained in the first step, transferring the centrifugal supernatant into the tube, and uniformly blowing and sucking the centrifugal supernatant by using a suction head; pipetting 600. mu.l of the mixture, transferring the mixture to a nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute; discarding the filtrate, placing the nucleic acid purification column back into a 2ml centrifuge tube, transferring the remaining mixture to the nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute;
step three, washing and deproteinizing: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 500. mu.l of washing solution 1, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step four, washing and desalting: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 700. mu.l of washing solution 2, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step five, eluting RNA: discarding the filtrate, putting the nucleic acid purification column back into a 2ml centrifuge tube, and centrifuging at 14,000rpm for 1 minute; discarding the filtrate and centrifuge tube, placing the nucleic acid purification column into a 1.5ml of centrifuge tube without RNase, adding 50 μ l of RNase-Free Water into the center of the membrane of the purification column, tightly covering the tube cover, standing at room temperature for 1 minute, and centrifuging at 12,000rpm for 30 seconds; the column was discarded and the eluted RNA was used immediately for molecular biology experiments or stored at-80 ℃ until use.
Example 4
RNA extraction was performed on 3 parts of fresh human anticoagulated whole blood according to the lysate formulation of example 3, comprising the following steps:
step one, whole blood lysis: adding 1ml of the rapid lysis solution into a 2ml centrifuge tube, adding 500 mu l of unfrozen human anticoagulated whole blood, covering a tube cover, carrying out vortex oscillation for 30 seconds, and centrifuging at 13,000rpm for 10 minutes;
step two, transferring 700 mu l of supernatant into a new 1.5ml centrifuge tube, storing for one week at the temperature of minus 20 ℃, and then entering the step three;
step three, adding 400 mu l of absolute ethyl alcohol into the 1.5ml centrifuge tube containing the supernatant, and uniformly mixing by blowing and sucking with a suction head; pipetting 600. mu.l of the mixture, transferring the mixture to a nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute; discarding the filtrate, placing the nucleic acid purification column back into a 2ml centrifuge tube, transferring the remaining mixture to the nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute;
step four, washing and deproteinizing: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 500. mu.l of washing solution 1, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step five, washing and desalting: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 700. mu.l of washing solution 2, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step six, eluting RNA: discarding the filtrate, putting the nucleic acid purification column back into a 2ml centrifuge tube, and centrifuging at 14,000rpm for 1 minute; discarding the filtrate and centrifuge tube, placing the nucleic acid purification column into a 1.5ml of centrifuge tube without RNase, adding 50 μ l of RNase-Free Water into the center of the membrane of the purification column, tightly covering the tube cover, standing at room temperature for 1 minute, and centrifuging at 12,000rpm for 30 seconds; the column was discarded and the eluted RNA was used immediately for molecular biology experiments or stored at-80 ℃ until use.
The results of agarose gel electrophoresis of the extracted RNA are shown in FIG. 4.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (7)
1. A whole blood RNA rapid lysis solution is characterized in that every 1000ml volume is composed of the following components by mass/volume:
guanidine thiocyanate, 0.2M-4M;
ammonium thiocyanate, 0.2M-3M;
anhydrous sodium acetate, 0.01M-0.2M;
glacial acetic acid, 0.05M-0.3M;
0.1-2% (W/V) of sodium lauroyl sarcosinate;
sodium chloride, 0.02M-0.3M;
1% -15% (V/V) of glycerol;
water saturated phenol solution, 10% -75% (V/V);
the rest is DEPC treated water.
2. The application of the whole blood RNA rapid lysate in the whole blood column method for extracting RNA according to claim 1.
3. Use according to claim 3, characterized in that it comprises the following steps:
step one, whole blood lysis: adding 1ml of the rapid lysis solution into a 2ml centrifuge tube, adding 500 mu l of fresh whole blood or bone marrow, covering a tube cap, carrying out vortex oscillation for 30 seconds, and centrifuging at 13,000rpm for 10 minutes;
step two, passing the RNA-containing supernatant through a purification column: adding 400 mu l of absolute ethyl alcohol into a new 1.5ml centrifuge tube, sucking 700 mu l of the centrifugal supernatant obtained in the first step, transferring the centrifugal supernatant into the tube, and uniformly blowing and sucking the centrifugal supernatant by using a suction head; pipetting 600. mu.l of the mixture, transferring the mixture to a nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute; discarding the filtrate, placing the nucleic acid purification column back into a 2ml centrifuge tube, transferring the remaining mixture to the nucleic acid purification column, tightly covering the tube cap, and centrifuging at 13,000rpm for 1 minute;
step three, washing and deproteinizing: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 500. mu.l of washing solution 1, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step four, washing and desalting: discarding the filtrate, putting the nucleic acid purification column back to a 2ml centrifuge tube, adding 700. mu.l of washing solution 2, tightly covering the tube cover, and centrifuging at 13,000rpm for 1 minute;
step five, eluting RNA: discarding the filtrate, putting the nucleic acid purification column back into a 2ml centrifuge tube, and centrifuging at 14,000rpm for 1 minute; discarding the filtrate and centrifuge tube, placing the nucleic acid purification column into a 1.5ml of centrifuge tube without RNase, adding 50 μ l of RNase-Free Water into the center of the membrane of the purification column, tightly covering the tube cover, standing at room temperature for 1 minute, and centrifuging at 12,000rpm for 30 seconds; the column was discarded and the eluted RNA was used immediately for molecular biology experiments or stored at-80 ℃ until use.
4. The use of claim 3, wherein the nucleic acid purification column is coated with SiO2Silica gel adsorption column of the material.
5. The use according to claim 3, wherein the lotion 1 is formulated with guanidinium thiocyanate 0.5M-4M, Tris-HCl (pH6.0-pH8.0)10mM-100mM, EDTA-2Na (pH8.0)10mM-50mM, and absolute ethanol 40% -60%.
6. The use according to claim 3, wherein the washing solution 2 is Tris-HCl (pH6.0-pH8.0)10mM-100mM, sodium chloride 20mM-300mM, and absolute ethanol 60% -80%.
7. The use according to claim 3, wherein the RNase-Free Water is 0.1% DEPC treated Water.
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| CN112708618A (en) * | 2020-12-30 | 2021-04-27 | 广州博江生物科技有限公司 | Lysate for quickly lysing cells, viruses and bacteria to extract nucleic acid |
| CN112725334A (en) * | 2021-02-23 | 2021-04-30 | 山东思科捷生物技术有限公司 | Cell RNA rapid extraction kit and RNA extraction method |
| CN114908081A (en) * | 2022-04-29 | 2022-08-16 | 杭州新景生物试剂开发有限公司 | Nucleic acid extraction reagent, kit thereof and method for rapidly extracting, separating and purifying nucleic acid |
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